Monoclonal antibody therapy is a large and growing treatment modality in medicine (Glennie et al. (2000) Immunol Today 21:403-10). There are more than twenty FDA-approved monoclonal antibody therapies, with many more currently in clinical trials. Antibody therapy directed against soluble factors (such as vascular endothelial growth factor or tumor necrosis factor, e.g.), aims simply to reduce the free ligand concentration by immunocomplex formation. In contrast, when antibody therapy is directed at cell surface antigens (as is often the case in anti-neoplastic immunotherapy), the goal is often the removal of the cell itself. The therapeutic antibody may induce apoptosis directly (Shan et al. (1998) Blood 91:1644-52; Shan (2000) Cancer Immunol Immunother 48:673-83), but more often it must recruit the patient's immune system to destroy the target cell. See, Reff et al. (1994) Blood 83:435-45; Idusogie et al. (2000) J Immunol 164:4178-84; Golay et al. (2000) Blood 95:3900-8; Harjunpaa et al. (2000) Scand J Immunol 51:634-41; Anderson et al. (1997) Biochem Soc Trans 25, 705-8; Clynes et al. (1998) Proc Natl Acad Sci USA 95:652-6; Clynes et al. (2000) Nat Med 6: 443-6; Sampson et al. (2000) Proc Natl Acad Sci USA 97:7503-8.
There are two main mechanisms by which the antibody-activated immune system can destroy offending cells: complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). ADCC is an immune response generated primarily by natural killer (NK) cells against antibody-coated targets. See, Lewis et al. (1993) Cancer Immunol Immunother 37:255-63. In ADCC, NK cells recognize the constant (Fc) region of antibodies primarily via interaction with the NK cell's FcγRIII receptor. The NK cells then deposit performs and granzymes on the target cell surface inducing cell lysis and apoptosis, respectively. The Fc-FcγRIII interaction is extremely sensitive to Fc glycosylation. Aglycosylated immunoglobulins fail to bind Fc receptors. See, Leatherbarrow et al. (1985) Mol Immunol 22:407-15 (1985); Walker et al. (1989) Biochem J 259:347-53 (1989); Leader et al. (1991) Immunology 72:481-5. In addition, fucosylation of the carbohydrate chain attached to Asn297 of the Fc region inhibits binding to FcγRIII and reduces in vitro ADCC activity. See, Shields et al. (2002) J Biol Chem 277:26733-40; Shinkawa et al. (2003) J Biol Chem 278:3466-73; Niwa et al. (2004) Cancer Res 64:2127-33.
The majority of mammalian immunoglobulins are fucosylated, including those produced in Chinese hamster ovary cells (CHO cells, Cricetulus griseus). Jefferis et al. (1990) Biochem J 268:529-37; Hamako et al. (1993) Comp Biochem Physiol B 106:949-54; Raju et al. (2000) Glycobiology 10:477-86. Fucose attachment to the Fc core region is via an α-1,6 linkage generated by the α1,6 fucosyltransferase (Fut8) protein, apparently the sole α-1,6 fucosyltransferase in mammalian cells. Oriol et al. (1999) Glycobiology 9:323-34; Costache et al. (1997) J Biol Chem 272:29721-8. Disruption of the FUT8 gene in CHO cells eliminated core fucosylation of antibodies and increased ADCC by around 100-fold. See, Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). However, such conventional gene disruption by homologous recombination is typically a laborious process. This was particularly true in the case of C. griseus FUT8, as approximately 120,000 clonal cell lines were screened to discover three healthy FUT8−/−clones (Yamane-Ohnuki et al. (2004), supra).
Thus, there remains a need for cells lines in which Fut8 expression is partially or fully inactivated. Site-specific cleavage of genomic loci offers an efficient supplement and/or alternative to conventional homologous recombination. Creation of a double-strand break (DSB) increases the frequency of homologous recombination at the targeted locus more than 1000-fold. More simply, the inaccurate repair of a site-specific DSB by non-homologous end joining (NHEJ) can also result in gene disruption. Creation of two such DSBs can result in deletion of arbitrarily large regions. The modular DNA recognition preferences of zinc fingers protein allows for the rational design of site-specific multi-finger DNA binding proteins. Fusion of the nuclease domain from the Type II restriction enzyme Fok I to site-specific zinc finger proteins allows for the creation of site-specific nucleases. See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and International Publication WO 07/014275, the disclosures of which are incorporated by reference in their entireties for all purposes.